Presbycusis, or ARHI, was characterized histopathologically by Schuknecht and Gacek (30
) in 1993. The two major types, sensory and neural, involve varying degrees of hair cell and cochlear neuronal cell loss and can often be found together. Loss of cochlear neurons is the most consistent finding in the aging ear. Cochlear neuronal counts can be as high as 40 000 in the first decade of life and decrease by more than 50% by the ninth decade in affected individuals (31
). The genetic mechanism underlying this age-related loss of approximately 2100 neurons per decade is not understood. The current study provides convincing genetic evidence that variation in a metabotropic glutamate receptor (GRM7) plays a role in ARHI. This includes data obtained in the Finnish population identifying two significant SNPs in GRM7 and significant data obtained in non-Finnish European subjects that replicated in an independent European replication group.
We believe that the GRM7 association is genuine for several reasons. First, we were able to replicate this association in a European replication group. The size of the replication group was small, and it is known that a replication group of smaller size than the original group used for association testing may lead to a lack of power for replication, and subsequent non-replication of results (known as the winner's curse) (32
). It is therefore possible that we have missed replication of other genuine results in our replication step. However, the result for GRM7 was significant. A second strong argument is that we have found association for GRM7 in the Finnish population, independently from the (replicated) association found in the non-Finnish European population. It must be noted that the association in the Finnish group was found in a different region of the gene than the association in the European group. GRM7 is a large gene, and the European and the Finnish association signals are approximately 300 kb apart. As the Finnish are known to be genetically distinct from the rest of Europe, possibly due to the documented historical population bottleneck (33
), this finding may be the result of allelic heterogeneity at the GRM7 locus. Allelic heterogeneity is frequently mentioned as a factor leading to the inability to replicate the exact SNP discovered and sometimes as the cause of lack of replication at an entire genetic locus. Some recent examples include reports on associations for rheumatoid arthritis (34
) and bipolar disorder (35
). However, allelic heterogeneity may not be the sole reason for replication of the association signal in a different region of the GRM7 gene. It is possible that the rs11928865 SNP exerts a smaller effect in the Finnish population or, if the effect size is similar, that the disease-associated allele frequency is small in Finland and therefore we may have been underpowered to detect the effect at that specific GRM7 SNP. A similar finding regarding disease allele frequency was discussed in a recent article (36
). Population specific differences in allele frequency of putative interacting SNPs could also lead to these findings. If the interacting locus is present at an altered allele frequency, there will be a large decrease in power to detect an association (37
). Lastly, we cannot rule out the possibility that our observation is a false positive association.
Individual genotyping of our large sample set was cost-prohibitive at the time this study was initiated; therefore, we based our GWAS on a pooling approach. It is important to recognize the advantages, as well as the disadvantages of such an approach, and we took several measures to overcome some of the limitations of pooled association experiments. First, to control for error, each pool was created de novo in triplicate and allelotyped independently on three separate arrays. Second, we analyzed our data in perhaps an overly strict fashion permitting only those SNPs with alleles associated in the same manner across all populations through to the next stage of analysis. Thirdly, before investigating the top most hits in our replication cohort, we individually genotyped a selected list of SNPs (detailed above) using an entirely different chemistry to provide further evidence that our initial findings were not due to pooling and/or microarray artifacts. This validation step indeed confirmed that the pooling approach led to reliable results as we confirmed the association of more than 35 and 90% of the selected SNPs in the European and Finnish samples, respectively.
The top 252 SNPs identified in the European sample group and the top 177 SNPs from the Finnish sample group did not contain any SNP from the three genes that previously had been implicated in ARHI: NAT2
) and GRHL2
). One possibility explaining this seeming non-replication is that the previously reported associations may have been spurious. However, this is unlikely as all three reported associations had previously been replicated in some way. In our opinion, the reason for not finding NAT2
SNPs among the top SNPs of the pooling experiment is possibly due to the lack of sensitivity of the pooling approach in combination with the large number of SNPs that we screened in the current study (500 K) compared to only a few dozen in candidate gene association studies. As a GWAS will pay a much larger multiple testing cost, less significant associations will always be ignored and only top-ranking SNPs will be considered further.
Metabotropic glutamate receptors (mGluRs) are activated by L-glutamate, the primary excitory amino acid neurotransmitter in the mammalian central nervous system (CNS). mGluRs are G-protein coupled receptors (GPCRs) functioning via secondary messenger pathways to modulate neuronal excitability and synaptic efficacy. The general structure of the metabotropic receptors consists of a glutamate binding site, a cystein-rich region, a seven transmembrane domain and an intracellular C-terminal region. To date, eight subtypes of mGluRs have been identified. GRM7 (mGluR7) is a member of the mGluR III group. mGluR7 is negatively coupled to the enzyme adenylate cyclase, and its activation results in reduced levels of cAMP. Its expression is temporally and spatially regulated in the human fetal brain. It is expressed at much lower levels in the adult brain in the hippocampus, hypothalamus and thalamus (38
). In general, the activation of presynaptic mGluRs, such as class III mGluRs, has been found to reduce transmitter release from synapses in many brain regions (39
). The mGluRs have diverse roles in the forms of synaptic plasticity that are believed to be involved in learning and memory in vertebrates. As such, mGluRs have attracted attention since their discovery as putative targets for many CNS indications, including anxiety, pain, neuroprotection, epilepsy, Parkinson's disease and cognitive disorders. mGluR7 has become particularly interesting since it was shown that its targeted deletion in mice results in several interesting neurological phenotypes including altered amygdala-dependent conditioned fear and aversion responses and reduced anxiety- and stress-related behaviors (40
To date, little is known about the physiological roles of the metabotropic glutamate receptors in the human inner ear. The mGluRI agonist DHPG produced a pronounced increase in afferent auditory nerve firing in vivo
, suggesting a role for metabotropic glutamate receptors in the mammalian inner ear (42
). Relatively recently, L-glutamate and ionotropic receptor subunits were detected by immunocytochemistry in human cochlear sections (43
), while mRNA of metabotrophic receptors group I (mGluRI) was detected in the mammalian cochlea and spiral ganglion (44
). Glutamate is known to be the principle neurotransmitter coupling the mechanoelectrical transduction of sound impulses between the inner hair cells and the auditory afferent neurons of the inner ear (47
). This was further confirmed by the fact that mice lacking Orphan Glutamate Receptor δ1 Subunit
(GluRδ1), which is normally expressed robustly in the neurons of the hippocampus, cochlear inner hair cells, outer hair cells and spiral ganglion cells, show high frequency sensorineural hearing loss (49
). The current study demonstrates robust mGluR7 expression in inner and outer hair cells and spiral ganglion neurons.
High concentrations of glutamate are known to be neurotoxic, because of its excitatory properties. Glutamate toxicity has been implicated in several forms of progressive hearing loss, including noise induced hearing loss and ARHI (50
). The results of our study implicate that GRM7 variants possibly act on ARHI through excitotoxicity. Because mGluR7 reduces the release of glutamate, we hypothesize that the causative allele of GRM7 alters synaptic autoregulation of glutamate in the synaptic cleft of the sensory cells of the inner hair cells and the auditory neurons, leading to higher levels of glutamate in the cleft over time with subsequent excitotoxic neuronal and/or sensory cell death. Recent support for glutamate toxicity in the ear comes from studies in cultured spiral ganglion explants. In a recent study, cultured spiral ganglion explants were incubated with high concentrations of glutamate (51
). The authors demonstrated concentration-dependent glutamate induced apoptosis of these explants that could be selectively blocked by a caspase-3 inhibitor.
GPCRs are the most successful class of drug targets to date. The identification of N,N′-dibenzhydrylethane-1,2-diamine dihydrochloride (AMN082), a highly selective agonist for mGluR7, has provided a valuable tool for studying the role of mGluR7 in stress-related CNS disorders (52
). AMN082 is orally active and has been shown to traverse the blood-brain barrier. Studies of hearing in aging mGluR7 deficient mice are currently underway and will provide invaluable initial insights into the molecular pathology of ARHI and a potential model for developing drug therapies.
In conclusion, the current pooling GWAS has provided convincing evidence that variation in GRM7 is associated with ARHI. Fine mapping has positioned the causative allele in the European population in a 150 kb region surrounding GRM7 exon 2. While the causative allele remains to be identified, this finding suggests a possible role for glutamate toxicity in the pathophysiology of the disease.